Borbat Petr P, McHaourab Hassane S, Freed Jack H
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
J Am Chem Soc. 2002 May 15;124(19):5304-14. doi: 10.1021/ja020040y.
We report the use of a novel pulsed ESR technique for distance measurement, based on the detection of double quantum coherence (DQC), which yields high quality dipolar spectra, to significantly extend the range of measurable distances in proteins using nitroxide spin-labels. Eight T4 lysozyme (T4L) mutants, doubly labeled with methanethiosulfonate spin-label (MTSSL), have been studied using DQC-ESR at 9 and 17 GHz. The distances span the range from 20 A for the 65/76 mutant to 47 A for the 61/135 mutant. The high quality of the dipolar spectra also allows the determination of the distance distributions, the width of which can be used to set upper and lower bounds in future computational strategy. It is also demonstrated that the shape of these distributions can reveal the presence of multiple conformations of the spin-label, an issue of critical relevance to the structural interpretation of the distances. The distances and distributions found in this study are readily rationalized in terms of the known crystal structure, the characteristic conformers of the nitroxide side chains, and molecular modeling. This study sets the stage for the use of DQC-ESR for determining the tertiary structure of large proteins with just a small number of long-distance constraints.
我们报道了一种基于双量子相干（DQC）检测的新型脉冲电子顺磁共振（ESR）技术用于距离测量，该技术可产生高质量的偶极光谱，从而显著扩展了使用氮氧化物自旋标记测量蛋白质中可测距离的范围。我们使用9 GHz和17 GHz的DQC-ESR对八个用甲硫基磺酸盐自旋标记（MTSSL）进行双重标记的T4溶菌酶（T4L）突变体进行了研究。距离范围从65/76突变体的20 Å到61/135突变体的47 Å。偶极光谱的高质量还允许确定距离分布，其宽度可用于在未来的计算策略中设定上限和下限。还证明了这些分布的形状可以揭示自旋标记的多种构象的存在，这一问题与距离的结构解释密切相关。根据已知的晶体结构、氮氧化物侧链的特征构象和分子建模，本研究中发现的距离和分布很容易得到合理的解释。这项研究为使用DQC-ESR仅通过少量长距离约束来确定大蛋白质的三级结构奠定了基础。
